1. Field of the Invention
The present invention relates to baculovirus-expressed influenza antigens, in particular, to the influenza A membrane protein, M2 expressed from Autographa Californica nuclear polyhedrosis virus (AcNPV). The invention further relates to immunoassays for the detection of influenza viral infections and to the use of such protein in vaccines against influenza A.
2. Background Information
The M2-protein of influenza A viruses is a membrane-spanning protein. It is found within membranes of virus-infected cells (R.A. Lamb and P.W. Choppin Virology 112:729-737 (1981) ; R.A. Lamb et al, Cell 40:627-633 (1985)). A small number of M2 proteins are also present in virus particles (S.L. Zebedee and R.A. Lamb J. Virol. 62:2762-2772 (1988)). Mutations occur in the transmembrane region of M2 protein present in viruses selected in vitro or in vivo to be resistant to the anti-viral agents amantadine and rimantadine (A.J. Hay et al, EMBO J. 4:3021-3024 (1985); W.J. Bean et al, J. Infect. Dis. 159:1050-1056 (1989)). One model for the function of M2 protein is that it possess ion-channel activity, which is inhibited by amantadine-like agents (R.J. Sugrue et al, EMBO J. 9:3469-3476, 1990; R.I. Sugrue et al, Virology 180:617-624 (1991). Amantadine-like agents include amantadine and various N-alkyl derivatives of amantadine which inhibit neuromuscular transmission by interacting with the ion channel of the nicotinic acetylcholine receptor and competitively inhibit the binding of other channel blockers, phencyclidine and histrionicotoxin, to the receptor. Although under some circumstances amantadine may indirectly interfere with the correct processing of the cleaved hemagglutinin of the Rostock strain of Fowl Plague Virus (R.J. Sugrue et al, EMBO J. 9:3469-3476 (1990)), it is a general rule that amantadine-like agents inhibit an early event in the replication of influenza A viruses, which occurs prior to transcription and translation of the genome of infecting virions (Hay et al, (1985); A.J. Hay and M.C. Zambon, Multiple actions of amantadine against influenza viruses. In Becker Y. (ed) Antiviral drugs and interferon: the molecular basis of their activity. Martinus Niihoff Publishing, Boston MA, pp. 301-315, (1984)) including the Rostock virus. Hence, even the small number of M2 proteins within virus particles are presumably involved in the early event blocked by amantadine. However, direct evidence about the function of M2 protein or its interaction with amantadine is lacking.
Since the M2 protein is conserved among various strains of influenza A virus, it may have potential for use as an influenza vaccine. It has recently been demonstrated that mice receiving passively transferred monoclonal antibody to M2 had lower liters of influenza virus in their lungs after intranasal challenge with live influenza virus (J. Treanor et al, J. Virol. 64:1375-1377 (1990)).
To facilitate structure-function studies of M2 protein, as well as to develop reagents needed for immunological studies, the present invention provides, in one particular aspect, the M2 gene of influenza A virus cloned into a recombinant baculovirus allowing its expression in insect cells.
Viral antigens produced by recombinant DNA expression systems can provide an inexhaustible source of chemically defined material for use in serodiagnostic assays, experimental vaccines, and fundamental research. These techniques also eliminate the costs and potential hazards in the large-scale cultivation of pathogenic viruses. For example, the use of baculovirus-expressed Hantaan virus nucleoprotein as a diagnostic antigen has been reported recently (Schmaljohn et al, Journal of General Virology 69:777-786 (1988)).
The recently developed eucaryotic expression system using recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV) , should be useful for producing antigens for immunoassays for the serologic diagnosis of viral infections (Luckow et al, Bio-technology 6:47-55 (1987) Infection of insect cells (Spodoptera frugiperda) with such recombinant baculoviruses allows for the production of large amounts of antigen (R.D. Possee, Virus Research 5:43-59 (1986)). In addition, the baculovirus system has other important advantages over the commonly used methods of producing viral antigens. For example, with the baculovirus system the viral antigens are produced in cells that do not contain antigens that cross-react with antibodies in most human serum. Therefore, the purification of the antigen that is required for proteins expressed in bacterial and yeast expression systems may not be required. Baculoviruses do not infect humans and can therefore be safely handled in large quantities.